In the second year of this project, we have executed the Materials Transfer Agreements necessary for us to obtain the published chordoma cell lines, including a new cell line UCH-2. We have tested the two known chordoma cell lines for colony formation, which is a prerequisite for testing the effect of ionizing radiation. Unfortunately, the cell line does not form adequate colonies. A third (derivative of UCH-1) cell line promised as a line that forms colonies, was found not to be adequate for our purposes. In both cell lines, using western blotting and immunoprecipitation, we have confirmed high-level expression of human T. Experiments have shown that chordoma cell line proliferation is significantly slowed by anti-T shRNA treatment. Appropriate assays confirm the downregulation of T mRNA and protein. cDNA microarray analysis is to compare cell lines treated with anti-T shRNA with control shRNA treated cell lines are currently underway and is expected to be completed within a month. Furthermore, the baseline microRNA environment of chordoma cells has been analyzed. That for cell lines downregulated for T by shRNA will soon be completed as well. Finally, we have also identified microRNA's that are altered in their expression after ionizing irradiation treatment of chordoma cell lines. cDNA microarray analysis of similar experiments are under way.